Functionalized nanopipette biosensor

ABSTRACT

Disclosed are methods and devices for biomolecular detection, comprising a nanopipette, exemplified as a hollow inert, non-biological structure with a conical tip opening of nanoscale dimensions, suitable for holding an electrolyte solution which may contain an analyte such as a protein biomolecule to be detected as it is passed through the tip opening. Biomolecules are detected by specific reaction with peptide ligands chemically immobilized in the vicinity of the tip. Analytes which bind to the ligands cause a detectible change in ionic current. A sensitive detection circuit, using a feedback amplifier circuit, and alternating voltages is further disclosed. Detection of IL-10 at a concentration of 4ng/ml is also disclosed, as is detection of VEGF.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/603,134 filed Jan. 22, 2015, which is acontinuation of U.S. patent application Ser. No. 12/435,056 filed May 4,2009, now U.S. Pat. No. 8,940,142 issued Jan. 27, 2015, which claimspriority from U.S. Provisional Patent Application No. 61/126,644, filedMay 5, 2008, the disclosures of which are hereby incorporated byreference in their entireties.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with government support under NIH GrantP01-HG000205 and NSF Grant DB10830141. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to the field of biomolecule sensing andsensors, the sensors having channels with tips having nanoscaleopenings.

Related Art

Presented below is background information on certain aspects of thepresent invention as they may relate to technical features referred toin the detailed description, but not necessarily described in detail.That is, certain components of the present invention may be described ingreater detail in the materials discussed below. The discussion belowshould not be construed as an admission as to the relevance of theinformation to the claimed invention or the prior art effect of thematerial described.

Several groups are developing single-molecule detection methods using ananopore (Deamer and Akeson, 2000; Deamer and Branton, 2002; Li et al.,2003). The first efforts in this field used an ion-channel protein toform the nanopore, and the ionic current through the pore was measured.The first reported success was the ability to detect singleoligonucleotides moving through the pore due to the blockage of ioniccurrent while the oligonucleotide traveled through the nanopore(Kasianowicz et al., 1996). This overcomes the common moleculardiagnostics drawback of requiring multiple copies of the analyte, sincesingle molecules were detectable. The ultimate goal of these efforts isto discriminate individual nucleotides in a DNA molecule based ondifferential blockage of the ionic current.

Specific Patents and Publications

Karhanek M., Kemp J. T., Pourmand N., Davis R. W. and Webb C. D, “SingleDNA molecule detection using nanopipettes and nanoparticles,” Nano Lett.2005 February; 5(2):403-7 discloses that single DNA molecules labeledwith nanoparticles can be detected by blockades of ionic current as theyare translocated through a nanopipette tip formed by a pulled glasscapillary. The disclosed set up uses a voltage clamp circuit, whichutilized a single detecting electrode in a bath to detectnanoparticle-DNA current block.

Ying et al., “The scanned nanopipette: a new tool for high resolutionbioimaging and controlled deposition of biomolecules,” Phys. Chem. Chem.Phys., 2005, 7, 2859-2866, DOI: 10.1039/b506743j, disclose a nanopipettewhich can also be used for controlled local voltage-driven applicationof reagents or biomolecules and this can be used for controlleddeposition and the local delivery of probes for mapping of specificspecies.

Umehara et al., “Current Rectification with Poly-L-lysine Coated Quartznanopipettes,” Nano Lett. 6(11):2486-2492 (2006) discloses currentresponses of noncoated and Poly-1-lysine coated nanopipettes using ananopipette in a bath solution.

Umehara et al., “Label-free biosensing with functionalized nanopipetteprobes,” Proc. Nat Acad. Sci. 106(12): 4611-4616 (Mar. 24, 2009),published after the provisional filing date, discloses certain aspectsof work described below and is specifically incorporated herein (alongwith other references cited here) for description desired for a fullerunderstanding of aspects of the present invention disclosed there.

BRIEF SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features andaspects of the present invention, nor does it imply that the inventionmust include all features and aspects discussed in this summary.

In certain aspects, the present invention comprises a device forspecific detection of an analyte in a sample containing electrolyte,said device comprising a nanopipette having a tip with a nanoscaleopening between an interior of the nanopipette and an external area forcontacting the sample, said nanopipette defining an internal volumecommunicating with the sample. The internal volume will containelectrolyte, and the sample will contain electrolyte. The purpose of theelectrolyte is to provide a source of ions for ionic current flow. Thenanoscale opening may be on the order of 10-100 nm, typically 50 nm, andthe opening is sized so that binding of an analyte in or near theopening will impede current flow, even if only one or a few moleculesare bound. Blocking by specific binding of an analyte causes a reductionof ionic current flow through the electrolyte solution. Thus, the tiphas chemically attached thereto, in the vicinity of the opening, apeptide-binding molecule specifically binding to the analyte in thesample. The chemical attachment preferably includes a covalent linkage,through a variety of pre-layers attached to the quartz nanopipette; as afinal step, a binding molecule, such as one comprising protein A, whichspecifically and tightly binds the Fc portion of antibodies, may beused. The device of the invention further comprises a first electrode,arranged to be in contact with electrolyte in the interior of thenanopipette. The electrolyte is part of, or is added to the sample; thesample inherently contains the analyte to be detected. The sample mayalso be modified to contain various controls, or analyte modifyingmaterials, although no analyte label is needed, and no inert or otherlarger particle is attached to the analyte molecules. Analyte in thevicinity of the opening is captured by the peptide binding ligand, asthe tip of the nanopipette is in contact with sample containing analyte.The first electrode, in the electrolyte interior of the nanopipette, isfurther arranged to be connected to an amplifier input in a currentdetecting circuit. The device further comprises a second electrode,arranged to be in contact with the electrolyte, and the second electrodeis exterior of the nanopipette, and further arranged for connection tothe current detecting circuit, whereby electrolyte in the interior ofthe nanopipette and electrolyte in the bath permits ionic current toflow between the electrodes and through the tip, said ionic currentbeing detectibly reduced when the tip is blocked by analyte.

As an example of use of the present device, a peptide binding ligandattached at the opening of the tip will bind specifically to thecorresponding receptor (e.g., antigen enzyme, small molecule ormetabolite). This technology can have a great impact in many areasincluding basic research, health care, environmental monitoring, andhomeland defense.

Thus the present invention, in certain aspects, may be used to detectand uniquely identify biological markers, pathogens, or contaminantswithout the need to label or pre-process them. They are identified bytheir differential blockage events of the ionic current while passingthrough a functionalized nanopipette. As an example, cancer biomarkerswhich may be present in serum are measured.

The present invention, in certain aspects, may also be used to detectvarious protein molecules and protein metabolites by detecting andanalyzing current blockage events resulting from binding to themolecules' counterparts (ligands) which are attached to the nanopipettetip. Blockage events can represent not only permanent binding events ofthe target molecule to the probe molecule but also conformationalchanges of the final molecular complex.

The present invention, in certain aspects, may also be used todistinguish between various molecules passing through the nanopipetteand creating characteristic blockage events. Statistical or patternrecognition analysis of these events produces parameters which are usedin identification of target molecules. For example, statistical analysisis described in Butler et al., “Ionic Current Blockades from DNA and RNAMolecules in the a-Hemolysin Nanopore,” Biophys J. 2007 Nov. 1; 93(9):3229-3240.

The present invention, in certain aspects, may also be used to usenanopipette tips coated with various agents to enable and improvedetection of various molecules. Coating of the nanopipette tip createsbetter resolution of the nanopipette by appropriate adjustment of itssize and surface chemistry for particular target molecules. Coating isalso used to enable and create conditions for surface attachment ofprobe molecules capturing target molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an apparatus containing functionalizedAxopatch voltage-clamp experiment diagram, showing two voltage channels,V_ch1 and V_ch2. In addition, two currents will flow through the tipregions of the nanopipettes, I-ch1, in the vicinity of tip 130, andI_ch2, in the vicinity of tip 132.

FIGS. 2A, 2B and 2C is a series of schematic drawings showing methodsused in attaching antibodies or other peptide ligands to a nanopipettesurface such that the ligands are in the vicinity of the tip opening.

FIG. 3 is a schematic diagram showing chemical functionalization of aglass surface, such as nanopipette tip, for attachment of peptideligands.

FIG. 4A and 4B is a set of graphs showing current traces from additionof IL 10 (FIG. 4A) and VEGF (FIG. 4B) to the device as illustrated inFIG. 1. The line data as presented in the following FIGS. 5-8 aresimplified in that only the peaks are shown; the peaks are connected topresent a single line representing a connected series of dots from thepeaks.

FIG. 5 is a graph showing nanopipette current response comparinganti-IL-10 IgG treated pipette with a control, anti-ferritin, showingcurrent changes detectible upon binding of IL-10 to antibodies on thenanopipette. Lines 402 and 408 are from antiferritin; line 404 shows acurrent reduction trace and line 406 shows a current increase traceresulting from anti-IL10 interaction with IL-10.

FIG. 6 is a graph of an experiment numbered 080122, using anti-VEGFantibody binding to VEGF. It shows lines connecting peaks and thecurrent change over time after adding VEGF.

FIG. 7 is a similar graph from experiment numbered 080205, which usedanti-IL10 antibody binding to IL-10. It showed a big change in thenegative ion current response and a small but significant change in thepositive ion current response for the anti-human IL-10 IgGfunctionalized nanopipette.

FIG. 8 is a graph showing results from a limit of detection experiment,numbered experiment 080224. Addition of analyte at differentconcentrations is shown.

FIG. 9 is a schematic drawing showing changes in ionic flow that resultfrom the formation of an electrical double layer (EDL) on the innersurface of the nanopipette. On the left, ionic flow increase by anelectrical double layer (EDL) is shown, resulting in a large currentchange. On the right side, with positive voltage applied, there isillustrated ionic flow resistance by EDL, leading to a small currentchange. The circled + is positive surface charge. As a result of thesurface charge an electrical double layer (EDL) of Cl— is formed. As aresult the nanopipette biosensor is partially ion permselective and alarger change in current will be observed when negative voltage isapplied due to the EDL.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Overview

The present methods and devices are directed to nanopipettes which havebeen chemically modified in their tip areas to contain a peptide ligandwhich may be used for rapid and sensitive detection of antigens in asample. One application of this technology is a point-of-care diagnosticdevice that can test a patient's sample for the presence of specificknown pathogens and determine the proper course of treatment. This canalso be useful for current research applications that use ELISA orprotein microarrays for readout. The present methods do not require thatthe analyte be labeled or attached to a nanoparticle to increaseblocking ability. No pretreatment of the sample is required.Alternatively, an assay similar to ELISA could be performed with thissystem, except that the colorimetric or fluorescent readout would bereplaced with nanoparticle labels. Of course, the nanochannel (innerdiameter of the nanopipette) and size of the particles would be scaledaccordingly to result in the appropriate detectable signals. In thisexample, antibodies could be labeled with differently-sizednanoparticles. The bound and unbound antibodies would be detected by thelength of the ionic block, with a longer block resulting with anantibody bound to a large protein. A very quantitative ELISA-like assaywould result, since individual molecules would be analyzed.

An additional application would be the analysis of nanoparticlesyntheses. Several groups are developing processes to synthesizenanoparticles with different materials for many different applications(Sun and Murray, 2000; Peng et al., 2000; Puntes et al., 2001). Theanalysis of such syntheses is labor intensive, involving the imaging ofsynthesized products with an electron microscope. The electronmicrographs are then analyzed for determining the size and uniformity ofthe synthesis. The system proposed in this effort would rapidly analyzenanoparticles, with the size being determined by the amount of ioniccurrent blocked.

Devices according to the present invention may also involveprotein-based detection. The ability to perform sensitive, real-time,and cost effective proteome analysis is of crucial value in clinicaldiagnostics, academic research, and drug development. Described below isan electrical-based, nanopipette biosensor with the ability to carry outbiomolecule detection. The present immunoanalytical method based onantibody-antigen interaction has a specific nature and adaptability. Thesurface chemistry's proof-of-principal was made in a glass slide modelsystem and subsequently implemented to the nanopipette biosensor. Inrepeated voltage clamp experiments the nanopipette biosensors proved tospecifically detect antigen cancer marker molecules VEGF and IL-10, at aconcentration of 4 μg/mL. Nanopipette bio sensor limit of detectionremains unknown but it is believed to be <4 ng/mL.

Compared to ELISA, nanopipette biosensors could have several potentialadvantages. The use of enzymes and other detection agents are notnecessary with nanopipette biosensors, which make the technology lessexpensive and more sensitive. With nanopipette biosensors we measureantigen-antibody real-time binding events and because of the computerinterface, more comprehensive data for more than just quantitativeanalysis can be collected.

The fact that nanopipette biosensor detection is done in real-time couldmake it an attractive diagnostic tool since it might allow close toinstantaneously patient bedside analysis with little waiting time fordiagnostic results. With the nanopipette platform we have shown specificbinding on one pipette with successful negative control on another inthe same sample bath, hence nanopipette multiplexing could beimplemented. Verification of specific binding with different negativecontrols is also desirable. Of high interest is determination ofnanopipette biosensor limit of detection, both for clean target moleculesample and for target molecule in plasma. Also, to re-use thenanopipettes for other antibodies or more experiments is possible, giventhat one could alter the pH (elution buffer could be used) or rinse thenanopipettes in high salt buffers. This method has been proven to workin Protein A/G affinity chromatography.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. Generally, nomenclatures utilized inconnection with, and techniques of, cell and molecular biology andchemistry are those well known and commonly used in the art. Certainexperimental techniques, not specifically defined, are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. For purposes of theclarity, following terms are defined below.

The term “nanopipette” means a hollow self-supporting, inert,non-biological structure with a conical tip opening of nanoscale, i.e.,0.05 nm to about 500 nm, preferably about (+ or −20%) 50 nm. The hollowstructure may be glass or quartz, and is suitable for holding inside ofit a fluid which is passed through the tip opening. The interior of thenanopipette is selected or modified to minimize nonspecific binding ofanalyte. The interior is sized to allow insertion of an electrode thatcontacts solution in the nanopipette.

The term “ionic current” means an electric current which flows throughan electrolyte material, such as salts or buffer in solution in apolymer, etc., which provides an ionically conductive medium, as opposedto an electronic current flow such as electrons in a wire. The ioniccurrent is carried by a flow of charged ions, such as in an electrolytesolution.

The term “analyte” is used in a conventional sense and in conjunctionwith the phrase “analyte binding molecule,” or ligand (to the analyte).The term “analyte” is used herein broadly to refer to any substance tobe analyzed, detected, measured, or labeled. Examples of analytesinclude, but are not limited to: proteins, peptides, hormones, haptens,antigens, antibodies, receptors, enzymes, nucleic acids (DNA and RNA),polysaccharides, chemicals, polymers, viruses, prions, toxins, organicdrugs, inorganic drugs, allergens, pollutants and nanoscale combinationsthereof. It will be understood that detection of, for example, a cell,is typically carried out by detecting a particular component, such as acell-surface molecule, and that both the component and the bacteria as awhole can be described as the analyte.

As used herein a “peptide analyte binding molecule” or “ligand” broadlyencompasses any peptide reagent that highly preferentially binds to ananalyte or target of interest, relative to other analytes potentiallypresent in a sample. A target (analyte) and target-specific(analyte-specific) reagent are members of a binding pair, and eithermember of the pair can be used as the target-specific reagent in orderto selectively bind to the other member of the pair. Examples of targetand target-specific reagent pairs include, but are not limited to,antigen and antigen-specific antibody; hormone and hormone receptor;hapten and anti-hapten; biotin and avidin or steptavidin; enzyme andenzyme cofactor; and lectin and specific carbohydrate. The presentinvention employs peptide analyte binding molecules that are fixed tothe nanopipette by chemical linkages, as described below.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably to refer to an oligomer or polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical analogue of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers.

The term “antibody” refers, as is customary in the art, to a proteinconsisting of one or more polypeptides substantially encoded byimmunoglobulin genes or fragments of immunoglobulin genes. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin (antibody)structural unit is known to comprise a tetramer. Each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). TheN-terminus of each chain defines a variable region of about 100 to 110or more amino acids primarily responsible for antigen recognition. Theterms variable light chain (VL) and variable heavy chain (VH) refer tothese light and heavy chains respectively.

The term antibody as used herein includes antibody mimics and antibodyfragments. Thus, the term antibody, as used herein also includesantibody fragments either produced by the modification of wholeantibodies or synthesized de novo using recombinant DNA methodologies.Preferred antibodies include single chain antibodies, more preferablysingle chain Fv (scFv) antibodies in which a variable heavy and avariable light chain are joined together (directly or through a peptidelinker) to form a continuous polypeptide.

A single chain Fv (“scFv” or “scFv”) polypeptide is a covalently linkedVH:VL heterodimer which may be expressed from a nucleic acid includingVH- and VL-encoding sequences either joined directly or joined by apeptide-encoding linker. Huston, et al. (1988) Proc. Nat. Acad. Sci.USA, 85:5879-5883. A number of structures for converting the naturallyaggregated but chemically separated light and heavy polypeptide chainsfrom an antibody V region into an scFv molecule which will fold into athree dimensional structure substantially similar to the structure of anantigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405and 4,956,778.

The term “antibody polypeptide” means a polypeptide having immunologicalspecificity to discriminate between different epitopes and bind withhigh affinity to the cognate antigen, typically with a Kd=about 1×10⁻⁸M, or between 10⁴ M and 10⁻¹¹M. Such polypeptides may include the abovereferenced IgG antibodies and antibody fragments, as well as specificbinding peptides such as high affinity receptors, or other peptidesobtained e.g., by screening a phage display library for bindingspecifically to an analyte of interest, as is known in the art (See,e.g., U.S. Pat. No. 6,828,110, to Lee, et al., issued Dec. 7, 2004,entitled “Assays for detection of Bacillus anthracis,” describingpolypeptides that specifically bind to B. anthracis.)

The term “current detecting circuit” may comprise any sensitive devicefor detecting changes in current on the order of 1-10 picoamperes, basedon a baseline current of 10-1000 picoamperes. The term further refers toa circuit that is time responsive and relatively temperature independentor allow for changes in temperature to be compensated for. It shouldhave an input in a circuit where a known voltage is supplied. Sensitivedetecting circuits are known, including voltage clamp amplifiers andtransimpedance amplifiers. The term “voltage clamp” here refers tocircuits which utilize a differential amplifier having one inputconnected to a variable command voltage, another input connected to ameasured voltage, and a feedback circuit. The voltage clamp usesnegative feedback to maintain the system at the command voltage, whichin this case is a predetermined alternating signal, such as analternating voltage signal from a signal generator. The output currentfollows changes in the input voltage and small changes in current can bedetected.

The term “quartz” is used herein as a nanopipette media is a fusedsilica or amorphous quartz, which is less expensive than crystallinequartz. Crystalline quartz may, however, be utilized. Ceramics and glassceramics and borosilicate glasses may also be utilized but accuracy isnot as good as quartz. The term “quartz” is intended and defined toencompass that special material as well as applicable ceramics, glassceramics or borosilicate glasses. It should be noted that various typesof glass or quartz may be used in the present nanopipette fabrication. Aprimary consideration is the ability of the material to be drawn to anarrow diameter opening.

The term “electrolyte” is used herein to refer to a material thatcontains electrolyte solids, i.e., free ions. Typical ions includesodium, potassium, calcium, magnesium, chloride, phosphate andbicarbonate. Other ionic species may be used. The material willtypically be liquid, in that it will comprise the sample, containing theanalyte, and the ions in solution. The sample itself may be anelectrolyte, such as human plasma or other body fluids, water samplesand so on. The electrolyte should carry an ionic current; about10-100mM, preferably about 100 mM of positive and negative ionic speciesare thought to be required for this function. The present device mayemploy either the same or different electrolytes in the nanopipetteinterior and in the sample material.

The term “protein A” refers to a 40-60 kDa MSCRAMM surface proteinoriginally found in the cell wall of the bacteria Staphylococcus aureus.It is encoded by the spa gene and its regulation is controlled by DNAtopology, cellular osmolarity, and a two-component system calledAr1S-Ar1R. It has found use in biochemical research because of itsability to bind immunoglobulins. It binds proteins from many ofmammalian species, most notably IgG's. It binds with the Fc region ofimmunoglobulins through interaction with the heavy chain. In certainaspects, the present methods use a protein comprising protein A, meaninga fusion protein, but also including other protein constructs which havea high binding capacity for the Fc portion of immunoglobins. Furtherexemplary details may be found in EP0324867, “Fc-binding protein andstrain of producing the same.”

Generalized Method and Apparatus

Using finely drawn capillary like tubes, functionalized by chemicallinkage to antibodies or peptide-based antibody-like molecules, targetantigens are detected. The target antigens may be protein moleculeswhich are introduced into the nanopipette and pass a specific bindingmolecule attached to the nanopipette tip. If they bind permanently, apermanent blockage of the ionic current is detected. The permanentblockage event is positive identification of the target molecule. Inthis detection system, target protein molecules in a sample can beuniquely identified by relatively permanent binding (long lasting) tothe specific probe molecule attached to the nanopipette tip.

The primary target molecules used in the examples below were two cancerbiomarkers, VEGF and IL-10. Biomarkers are indicators of change inprotein expression related to disease and the progression of disease.Therefore they are commonly used for clinical diagnosis and analysis ofthe disease's stage. In addition, biomarkers help in the tailoring oftreatments for diagnosed individuals. However, the biomedical techniquesused for protein analysis today are not well developed and a new tool isneeded for rapid, multiplexed and accurate analysis of biomarkers.

Two different cancer biomarkers were used in this project; VascularEndothelial Growth Factor (VEGF) and interleukin-10 (IL-10). VEGF is agrowth factor protein inducing increased endothelial cell permeability,angiogenesis, and endothelial cell growth, thus promoting metastasis.Occurrence of over expression of VEGF has been found in many types ofcancers, for instance breast cancer and colorectal cancer, and iscorrelated with poor prognosis of survival in these cancer patients.IL-10 is an immunosuppressive and anti-inflammatory agent producedwithin the body, and it is also known as “human cytokine synthesisinhibitory factor” because it suppresses the production of cytokines.The correlation between cancer and IL-10 is somewhat ambiguous since itcan both serve as a growth factor for cancer cells and promote theinnate immune effector mechanisms leading to cancer cell destruction.Even though IL-10 over expression cannot be correlated to cancer patientsurvival prognosis, it is highly related to cancer and for this reasonit can be used as a biomarker in cancer diagnosis. Otherantigen-antibody combinations may be used; any specific protein-ligandinteraction may provide appropriate molecules for use in the presentmethods and devices. The present examples enable the development ofmultiplexed nanopipette technology into a diagnostic device with theability of real-time protein detection in complex samples, with greatersensitivity and specificity than existing techniques such as ELISA andsurface plasmon resonance sensors. A multiplexed nanopipette biosensorshould be able to make detections within small human sample volumes andat low target concentrations.

Once the surface chemistry proof-of-principle was established, theresultant surface chemistry was implemented on the nanopipettes. Voltageclamp type measurements were performed with functionalized nanopipettesas biosensors with the goal of proof-of-principle. Sensitivity,selectivity, and robustness of the nanopipette platform wereinvestigated involving two different cancer marker proteins.

As shown in FIG. 1, the present apparatus comprises functionalizednanopipettes having inserted therein electrodes and arranged to contactan analyte solution 118 and detection circuitry. In this case, twoamplifiers 102, 104 are used for detection of current blockage at thetip of the nanopipettes, because two nanopipette electrodes are used.The two working electrodes 114 are separated by an electrolyte gel 126exemplified below by agar. Two circuits are used, applying voltagesV_ch1 and V_ch2, one voltage supply to each pipette. The applied voltageto each of the nanopipettes result in currents I_ch1 and I_ch2, separatecurrent responses at the tips of the nanopipettes. The A/D converterconverts the analog voltage signal to numbers for data analysis andpresentation.

One benefit from the present nanopipette-based biosensors is that theyprovide an electrical-based detection method, and thus a simpler andfaster system; the assay involves no gels, fluorescent or radioactivelabels, dyes or beads. This makes nanopipette biosensors an easy to use,sensitive and cost efficient tool in diagnostics. In addition,nanopipette fabrication is a single-step relatively cheap process andthe fabricated nanopipettes are easy to tailor to different types ofexperiments and applications. Immunoanalytical methods are adaptablesince we can produce antibodies against almost any particular compound.

The electrolyte gel acts as a filter to allow electrolyte and thus ioniccurrent to pass through the materials (i.e., electrolyte ions such asK+, Cl−, etc.), while preventing analyte (e.g., IL-10) molecules frompassing between the two electrode containing materials. Changes inanalyte in the bath having the working electrode will not affect thereference electrode. Other filter materials besides agar can be used.These include actual membrane materials or other gel materials, such aspolyacrylamide or agarose gels. The electrolyte gel filter serves tominimize cross contamination when two different analytes are beingmeasured, that is, if nanopipettes 113 a,b are functionalized with twodifferent ligands to detect two different analytes in a single sample. Ahigh degree of multiplexing is possible with the present device; forexample, a positive control pipette with a ligand to a known analyte, anegative control pipette, with a ligand to an absent analyte, and tendifferent sample-testing nanopipettes could be clustered in a singlesupport, owing to the small size of the pipettes. Another advantage ofusing an electrolyte permeable gel is that the reference electrode 116does not see analyte and may be reused easily in different tests.

As As shown in FIG. 2A-C, antibodies (or other peptide ligands) areattached in the vicinity of the tip of the nanopipette 204. The tipvicinity may be regarded as being within the ring of the opening, or onthe inside or even the outside, within a distance of several moleculardiameters of the analyte to be detected. Antibodies may be attached tothe outside of the nanopipette as well as the inside, as long as thereare a number of antibodies near the tip. Antibodies should not beattached beyond 1 mm from the tip opening, preferably not beyond 0.5 mmof the tip opening. The antibodies are attached by direct chemicallinkage to the glass or quartz surface. The antibody at the tip willbind to an antigen, causing a detectible current blockage, provided thatthe right detection circuit is used. As shown at FIG. 2A (first step),protein molecules 206 will be contained in or extracted from the sampleto be tested. The specific binding molecules 202 will be attached to thenanopipette tip (second step; FIG. 2B). Chemistry for tightly bindingthe antibodies to the tip region is described below. FIG. 2C illustratesthe mechanism whereby a passing molecule 208 specific for the bindingmolecule will be detected upon its passage through the channel in thenanopipette (third step).

FIG. 3 shows a series of six steps in which a glass or quartznanopipette is chemically modified for covalent linkage of antibodyproteins (or other peptide binding molecules) to the surface. In step 1,the negatively charged glass slide/nanopipette (1.) is coated with amonolayer of poly-1-lysine (PLL) (2.) altering the charge and supplyingamine groups. Next, the surface is treated with a carboxylated polymersuch as polyacrylic acid or polymethacrylic acid, containing multiplecarboxyl groups (3.) and with the help of the crosslinker EDC/NHS (4.),(1-Ethyl-3 -(3 -dimethylaminopropyl)-carbodiimide/N-Hydroxysuccinimide)a linkage between the surface and added Protein A/G (5.) is formed. Asdescribed in detail below, the EDC/NHS has been previously used in othercontexts to provide an amide bond. In the final step, antibodies areimmobilized (6.) onto the glass slide surface. The underlying surfacechemistry was developed with glass slide experiments, was subsequentlyimplemented to the nanopipettes and is shown in FIG. 3. To enableprotein immobilization onto the glass pipette, there is a need to changeits negatively charged properties since most proteins have a netnegative charge. Numerous protocols had to be investigated in order toestablish what protocol would be implemented to the nanopipettebiosensors. Direct covalent coupling of the ligand peptide is preferred.This can be done by NHS linkage, silanization, or the method describedabove, which involves coating the glass with a PLL layer, a carboxyllayer, and then using EDC/NHS coupling. It is also desirable to couplethe antibodies through a protein that binds to the Fc portion of theantibody, such as protein A/G, as described below. Protein A/G is agenetically engineered protein that combines the IgG binding profiles ofboth Protein A and Protein G. Protein A/G is a gene fusion productsecreted from a non-pathogenic form of Bacillus. This geneticallyengineered Protein A/G is designed to contain four Fc binding domainsfrom Protein A and two from Protein G. Silanization may be used tocouple the peptide binding molecule to the nanopipette tip as described,e.g. in U.S. Pat. No. 5,077,210, issued Dec. 31, 1991. Various methodsfor coating inorganic substrates with silane films have been reviewed,Weetal H. H. (1976) Methods in Enzymology, Volume 44, 134-148, AcademicPress, New York, NY. Inorganic porous substrates coated with epoxysilane have been oxidized to produce aldehyde groups reacting directlywith antibodies, Sportsman, J. R. et al (1980) Anal. Chem. 52,2013-2018.Others, such as Sagiv U.S. Pat. No. 4,539,061, have establishedmultilayers of silanes deposited on silica. Proteins have further beenlinked to silane films on silica using glutaraldehyde. See U.S. Pat. No.4,478,946, Mandenius, C. F., et al (1984) Anal. Biochem. 137, 106-114,and Richards, F. M. et al (1968) J. Mol. Biol. 37, 231-233. Reactivecrosslinkers such as glutaraldehyde may bind to many residues and formmulti protein complexes which could interfere with protein function. Toavoid the use of glutaraldehyde, others have modified silica surfaceswith epoxy silanes and subsequently altered the silanes to have adihydroxy terminus, U.S. Pat. No. 4,562,157.

Protein A/G binds to all human IgG subclasses. In addition, it binds toIgA, IgE, IgM and to IgD but to a lesser extent to IgD. Thus, ProteinA/G may be a preferred ligand in tests for or tests using non-IgG classimmunoglobulins.

The basic surface chemistry used in the glass slide proof-of-principleand the nanopipettes has the following steps: First the slide is coatedwith poly-L-lysine (PLL) solution and then baked. The polycationicproperty of the PLL molecule allows it to bind electrostatically to thenegative quartz surface and gives the surface a better ability to bindto proteins by its amine groups. Secondly a carboxylated polymer isapplied. The carboxylated polymer, which is poly acrylic acid, becomes acarboxyl acid in aqueous solution and overcoats the PLL, thus cancelingthe positive charge. 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride/hydroxysulfosuccinimide/ N-hydroxysuccinimide (EDC/NHS) isused as a coupling agent for applied proteins. The EDC/NHS system isdescribed in the literature, e.g., in J. S. Daniels and N. Pourmand,“Label-free impedance biosensors: Opportunities and challenges,”Electroanalysis, vol. 19, no. 12, p. 1239, 2007.1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC orEDAC) is a zero-length crosslinking agent used to couple carboxyl groupsto primary amines. This crosslinker has been used in diverseapplications such as forming amide bonds in peptide synthesis, attachinghaptens to carrier proteins to form immunogens, labeling nucleic acidsthrough 5′ phosphate groups and creating amine-reactive NHS-esters ofbiomolecules. EDC reacts with a carboxyl to form an amine-reactiveO-acylisourea intermediate. If this intermediate does not encounter anamine, it will hydrolyze and regenerate the carboxyl group. In thepresence of N-hydroxysulfosuccinimide (Sulfo-NHS), EDC can be used toconvert carboxyl groups to amine-reactive Sulfo-NHS esters. This isaccomplished by mixing the EDC with a carboxyl containing molecule andadding Sulfo-NHS.

Thus, EDC is used here to couple carboxyl groups with proteins' aminegroups. When EDC reacts with a carboxyl it creates an amine-reactiveintermediate. If an amine bond is not created the intermediate ishydrolyzed and the carboxyl group is regenerated. In the presence ofNHS, the EDC intermediate is stabilized by the formation of a NHS esterand thereby NHS increases the efficiency of EDC. In order to minimizenon-specific binding, BSA was used as a blocking agent. The slide wasscanned at 532nm and the fluorescein dyed streptavidin bound to theimmobilized biotinylated antibodies was visualized. Non-biotinylatedanti-ferritin IgG was used as negative control.

Immobilization protocols including biotinylated and Cy3 conjugatedoligonucleotides and biotinylated BSA as probe molecules were tested,but proved to be less successful and were therefore not further pursuedexperimentally.

After adding a coupling layer of NHS esters, Protein A/G was added.Protein A/G is a genetically engineered, highly specific protein thatbinds to the Fc-region of IgA or IgG. Protein A/G is commerciallyavailable, e.g., from Pierce Protein Research Products. Protein A/G is agenetically-engineered protein that combines the IgG binding domains ofboth Protein A and Protein G. It is a gene fusion product expressed inE. coli. Protein A/G contains four Fc binding domains from Protein A andtwo from Protein G, resulting in a final mass of 50,460 daltons (40-45kDa by SDS-PAGE). Protein A/G is not as pH dependent as Protein A, butotherwise has the additive properties of Protein A and G. Protein A/Gwas utilized since it gives the surface the preferred alignment of theantibodies, e.g., active sites will be available to antigen binding,thus enhancing the effectiveness of the surface. After incubation overnight in cold room with protein A/G the surface is ready for antibodyimmobilization.

The target molecules passing through the nanopipette tip createcharacteristic blockage events of the ionic current. FIG. 5 showsresults from the addition of a detected analyte (IL-10), lines 404 and406, and a control analyte, ferritin, lines 402 and 408 (top and bottomlines).

Other experiments (FIGS. 6-8) further demonstrated experimentally thedetection of antigen-antibody interactions.

An important aspect of the present invention is the circuitry that isapplied to the functionalized nanopipettes. Electrodes are placed in thenanopipette and in an external eleoctrolyte solution. One externalelectrode is a reference electrode. An alternating voltage is applied tothe working electrode(s). This produces positive and negative ioniccurrents. Both are detected. A relatively low frequency alternatingvoltage is used. Computer data processing is used to measure a series ofpositive and negative current peaks and construct plots of these peaks,such as the lines shown in e.g., FIG. 5, 402-408. Thus, small changes incurrent flow can be detected and their significance as representingspecific molecular interaction at the nanopipette tip can be determined.

The present methods rely on specific protein interactions between theligands at the tip and the analyte. Exemplified are antibody-antigeninteractions. In certain embodiments, one may immobilize peptideantigens on the pipette tip and detect antibody analytes. Differentantibodies may be used, and will have differing avidity and affinity fortheir cognate antigens. In addition, different pipette tips will containdifferent numbers of immobilized antibodies in the tip region, due tomanufacturing variations. To this end, target molecules in a sample canbe further uniquely identified by statistical or pattern recognitionsignal analysis of blockage events while passing through the nanopipettetip. It is not necessary to rely on labeling the antigen to be detectedin the present methods. The analysis system will include the ability totake a clinical sample and test for the presence of specific molecules.In general, the task is to distinguish between various molecules passingthrough the nanopipette and creating characteristic blockage events.

EXAMPLES Example 1 Nanopipette Fabrication

Fabrication of “nanochannels” in glass capillaries results in what isreferred to as a “nanopipette.” Nanopipettes are fabricated usingthin-walled quartz capillaries with, for example, an initial innerdiameter of 0.7 mm. Different inner and outer capillary diameter can beused to achieve variations of shapes and sizes of nanopipettes. Thesepipettes are placed into a laser-based pipette puller (e.g., asavailable from Sutter instruments, Novato, Calif.), and the resulting“needle” can have a channel with smallest outer diameter of 10 nm at thetip (according to Sutter Instruments puller manual). Stanford hasproduced nanopipettes with outer diameter of approximately 50 nm at thetip, with the smallest observed outer diameter of 37 nm. The range oftested pipette outer diameters has been 37-82 nm, with an average of 56nm. The tip opening would be on this order of size. Although thepreferred material is quartz, other materials may be used. A variety ofmaterials consisting primarily of silicon dioxide are known and may beobtained commercially, e.g., from Technical Glass Products, Inc.,Painesville Twp., Ohio.

Ionic current consistent with the observed dimensions can be seen whenthe fabricated nanopipettes are filled with KCl solution. For example,with 100 mM KCl solution, the current measured in one pipette tip wasapproximately 200 pA with an applied voltage of 50 mV. This isconsistent with an inner tip diameter of approximately 40 nm (Equation1). Furthermore, the linear relationship between voltage and observedcurrent indicates that the nanochannel is not selective for the ions inthe solution and that there is no effective ionic gradient. This alsosuggests that the nanochannel resistivity for this solution is directlyrelated to the nanochannel dimensions.

The pipette tip geometry and corresponding resistance has been described(Sakmann & Neher, 1995, Ch. 21). In general, the resistance of thepipette is the sum of elementary resistances represented by slabs offluid with area A(x) and length dx:

$\begin{matrix}{R = {\int_{x\; 1}^{x\; 2}{\frac{\rho}{A(x)}{dx}}}} & \lbrack 1\rbrack\end{matrix}$

The nanopipettes have been fabricated, and ionic current through thesenanopipettes has been observed. Furthermore, blockages of current due tonanoparticles flowing through the pipette were also observed, withresults shown below.

The shape of the nanopipette is conical up to the close proximity of thetip. The very end of the tip (approximately the last 500 nm in length)has a conical angle that appears to be much steeper in cone angle at theend of the tip. The tip having a 52.2 nm opening was observed to haveabout a 100 nm radius of curvature at the tip.

A nanopipette tip may be examined by a scanning electron microscope forappropriate geometry. Typically, it will have an elongatedfrusto-conical “base” or “top,” tapering down into a narrower, finelydrawn tip, ending in a sharp tip, nearly pointed, with a small opening(e.g., ˜50 nm) at the end.

The nanopipette tip is approximately conical. A feature of the conicallyshaped nanopipette is that current change is focused to an area justinside of the nanopipette tip, thus creating a ‘sensing zone’ (See, Lee,S., et al., Electrophoretic capture and detection of nanoparticles atthe opening of a membrane pore using scanning electrochemicalmicroscopy. Anal Chem, 2004. 76(20): p. 6108-15.

The total resistance of the pipette is given by the sum of thecylindrical shank resistance and the resistance for the conical tip(Equation 2.1). (See, Sakmann, B. and Neher, E. Single ChannelRecording, Second Edition Chapter 21, pgs 638-639. Plenum Press, NewYork, 1995.) The resistance of the tip dominates since the radius of theshank, r_(s), is a lot bigger than the radius of the tip, r_(t).

Equation 2.1 shows total resistance, R, of a nanopipette. ρ is theelectrolyte resistivity, 1 is the length of the nanopore and θ is theangle of the nanopipette tip cone.

$\begin{matrix}{R = {\frac{\rho \; l}{\pi \cdot r_{s}^{2}} + {\frac{\rho \; {\cot \left( {\theta/2} \right)}}{\pi}\left( {\frac{1}{r_{t}} - \frac{1}{r_{s}}} \right)}}} & 2.1\end{matrix}$

This focused effect results in a very good detection system that isextremely sensitive to analyte molecules in the electrolyte, aphenomenon not seen in cylindrical nanopores.

In the present work, nanopipettes were fabricated from quartzcapillaries with filaments and with an outer diameter of 1.0 mm and aninner diameter of 0.70 mm (Sutter Instruments, Novato, Calif., USA). Thecapillary was placed in a P-2000 laser pipette puller machine (SutterInstruments, Novato, Calif., USA) pre-programmed to fabricatenanopipettes with the inner diameter of ˜50nm. Parameters used were:Heat=700, Fit=4, Vel=60, Del=150 and Put=192 and alterations of theseparameters can change the scale of the fabricated nanopipette tip. Withthe pre-set parameters mentioned nanopipettes tips with inner diametersranging from 37 to 82 nm have been produced and tested with the meansize being 56 nm (See, Karhanek, M., et al., “Single DNA moleculedetection using nanopipettes and nanoparticles,” Nano Lett, 2005. 5(2):p. 403-7.)

Example 2 Measurement Apparatus Setup and Procedure

The present apparatus is exemplified and illustrated in FIG. 1. Quartzglass nanopipettes 113 were fabricated and filled as described abovewith antigen solution 118. The antigen is in the nanopipette and theelectrolyte/analyte solution 120 into which the nanopipette is inserted.The antigen may be in a mixed and dilute form, and it will bespecifically identified from a complex mixture. Nanopipettes 113 a,b areplaced into a pipette holder (Axon Instruments, or Warner InstrumentsCorporation, Hamden, Conn.). The pipette holder is then attached to aMM-33 micromanipulator (Sutter Instruments Company) (not shown). Eachnanopipette 113 a,b contains an electrode 114 a,b that is connectedindividually to an Axopatch 200B amplifier (Axon Instruments, FosterCity, Calif.). Each electrode 114 is connected to a headstage 106, 108.This serves to reduce noise; the circuitry is also shielded in a faradaycage. Each headstage 106, 108 (Axopatch 200B and CV 203BU) was input toan operational amplifier at a negative input, where the positive inputis grounded or connected to a command voltage. The command voltage maybe set at different levels as is known in the field of voltage clampcircuitry. The amplifiers are output to an analog to digital converterfor data interpretation and storage by a computer. A digital to analogconverter connected to a computer is used to provide the commandvoltages.

The sample solution 120, consisting of 10-100 mM KCl, is placed into asmall beaker or on a hydrophobic surface resulting in a large drop ofapproximately 150 microliters. The pipette tips containing antibodies130, 132 are then carefully immersed into the solution and the testantigens are added to the solution next to the immersed pipette tip orinside of the pipette.

Ionic current is recorded using the Axopatch 200B amplifier involtage-clamp mode with signal filtering at 5-10 kHz bandwidth. HoldingCommand to set voltage commands in voltage clamp and current commands incurrent clamp, one makes a choice of three gain settings on thededicated current output based on nanopipette and working solutionconditions.

The signal is further digitized by an Axon Digidata 1320A digitizer withsampling frequencies from 10 kHz to 500 kHz. The data is recorded usingClampex 8 (Axon Instruments), and the same software is used for basicsignal analysis.

The nanopipette biosensor technology is based on an electrochemical cellconsisting of an electrolyte (working buffer; 100mM KCl, 2mM Phosphate)and two Ag/AgCl electrodes; one working electrode inserted to theelectrolyte filled nanopipette and one reference electrode 116. Theworking electrode and the reference electrode are in separate bathsconnected through electrolyte solution in an agar gel filled box. Theagar gel 126 keeps the baths separated and prevents analytecontamination. When voltage is applied to the circuit, ions will flowthrough the nanopipette opening, creating a steady current flux. Thisionic current is modified by changes in the nanopipette tip region andmay be blocked, partially or fully, by molecules translocating theopening. Nanopipette detection can be readily adapted for multiplexprotein detection by using several nanopipettes, each with a differentimmobilized probe protein, immersed in the same electrolyte bath. Afterapplying voltage to the system and upon addition of target protein tothe nanopipette bath, the nanopipette with the complementary probeprotein immobilized on its surface will differentiate itself againstother nanopipettes through a unique change in its current profile. Thisway the target protein or other antigen can be identified.

Apparatus Comprising Electrode Baths Separated by Electrolyte ContainingFilter Material Making the Agar Gel Box

200 mL buffer (100 mM KCl, 2 mM Phosphate) was microwaved together with2 g of agar powder until all the powder had dissolved. Then about 50 mLagar liquid was poured into an empty and clean pipette box and was putin cold room to stiffen. Agar gel filled plastic pipettes were also madeby pipetting hot aqueous agar gel up into the pipettes and then left toharden. Working buffer was poured onto the gel in the gel box, and thelevel of agar gel and buffer solution was marked.

Voltage Clamp Measurements

A voltage clamp device controls, or clamps, a nanopore potential at anylevel decided by the researcher. The voltage clamp technique was hereused to investigate how applied potential affects the ionic current flowthrough the nanopipette, and how changes in the nanopipette tip regionand in the nanopipette surface influence this ionic current.

The electric potential in the nanopipette tip is affected by the surfacecharge of the nanopipette. In the presence of a surface charge, anelectric double layer (EDL) of counter ions is recognized to be formedat the surface/electrolyte interface and causes the nanopipette tobecome ion permselective (See, FIG. 9 and Wei, C., A. J. Bard, and S. W.Feldberg, Current rectification at quartz nanopipette electrodes.Analytical Chemistry, 1997. 69(22): p. 4627).

A quartz nanopipette without any modifications has a negative surfacecharge; hence an EDL containing K⁺ ions will be formed with a KClelectrolyte solution. This phenomenon is reduced by higher electrolyteconcentrations but a 100 mM KCl electrolyte concentration, as used inthis project, gives rise to an EDL of about lnm, according to Wei et al.

There are two analyte detection principles with nanopipette biosensors;analyte or nanoparticle blockage of ion current leading to temporalreduction of ion current explained by Karhanek, et al., and change innanopipette surface ion conductivity induced by binding events. Theexperiments in the scope of this project made use of the latterdetection method.

The functionalized pipettes were filled with working buffer (100 mM KCl,2 mM Phosphate, pH=7.0, σ=12.1 [mS/cm]. Filling was performed firstthrough capillary forces by putting the shank side of the nanopipetteinto 40 μL buffer and visually seeing the tip being filled. Then theshank of the nanopipette was filled with working buffer with the help ofa syringe pipette. Finally the filled nanopipettes were mounted in thepipette holder (Axon Instruments Inc., Foster City, Calif., USA).Ag/AgCl working electrodes and a reference electrode were fabricatedwith silver wire and Clorox bleach and the working electrodes wereattached to the nanopipettes in the pipette holder. The nanopipettes andthe reference electrode were immersed into electrolyte baths in a 1%agar gel box. As shown in FIG. 1, a reference electrode 116 was placedin an electrolyte solution 122 which is physically separated from thepipette solution. It is separated by an electrolyte gel 126. Electrolytesolution is also contained in a bath into which two nanopipettes areinserted so that the tip openings are submerged. Each of the twopipettes contains a working electrode 114. In addition, a nanopipettemay be provided with a working electrode which is not functionalizedwith the antibody type molecule of interest, to serve as a negativecontrol. Each nanopipette has a port for addition of electrolyte and,optionally, analyte. The working electrodes 114, 114 a may have adifferent antibody 130, 132 immobilized in the vicinity of the tip. Oneantibody may be selected to give no binding to materials in the sample,and serve as a negative control. Alternatively, a nanopipette may beprovided with an antibody to a known marker added to the sample,providing a positive control. While only two electrodes/nanopipettes areillustrated in FIG. 1, it is understood that numerous functionalizednanopipettes may be simultaneously inserted into the sample.

Each working electrode 114 was connected to an Axopatch amplifierapplying input voltage and measuring output current. The analog signalwas low-pass filtered at 50 kHz with a four-pole Bessel filter. Theamplified, filtered signal was digitized at 250 kHz with a NI PCI-6014DAQ card (National Instruments, Austin, Tex.). Data acquisition wascontrolled with custom software written in LabWindows/CVI (NationalInstruments). Data analysis was implemented in MatLab (The MathWorks,Natick, Mass.). The Axopatch output is connected to the A/D converterthat is in turn connected to a computer with Clampfit data analysisprogram. A voltage cosine wave, +/−200 mV, was applied to the circuitand current response was measured. It can be seen in FIG. 1, that adigital to analog converter 112 is connected to the input(s) (positivein this example) of an amplifier such as shown at 103 and acts as asignal generator to provide a predetermined alternating voltage. Headstage amplifiers 106, 108 are connected between the electrodes and theamplifier 102 input. These are shielded to prevent noise, as by aFaraday cage. As described below, current changes being measured are onthe order of 3-20 picoamperes, and sensitive detection circuitry isneeded. Different analytes were added to the working electrolyte bathand current change was subsequently analyzed. The cosine wave couldalternatively be a sine wave. It is important to use a voltage whichalternates, so that rates of current change in different polarities canbe measured. A square wave or other shape could be used with propercorrections. The frequencies employed here have been found to be mostfavorable in a very low range, around one Hz (e.g. 0.5-10Hz). TheDigital to analog converter 112 is used as a signal generator to theinputs of the amplifier and is computer controlled to enable selectionof desired frequencies and amplitudes.

Example 3 Glass Slide Protocol for Determining Protein BindingConditions

A glass slide may be used for development or modification of a chemistryfor attaching a ligand to a nanopipette. In this example, a glasssurface is modified with a positive charge whereby a peptide bond may becreated with a protein ligand, namely the Fc portion of an antibody,e.g., biotinylated anti-human ferritin IgG. Attachment of the antibodymay be demonstrated by capture of fluorescence-coupled streptavidin.

A glass slide (Gold Seal Products, Portsmouth, N.H., USA) was cleaned ina sonicator for 30 minutes at room temperature. The slide was coated in0.01% poly-L-lysine (Electron Microscopy Sciences, Hatfield, Pa., USA),size 30,000-70,000 kDa, for 5 minutes at room temperature. Subsequently,the glass slide was baked in 120° C. for one hour to evaporate the waterbound with the PLL. Next the slide was incubated for 10 min incarboxylated polymer solution at room temperature. After washing theslide in water three times and blow drying it with argon gas it wasincubated with EDC/ NHS (Pierce Biotechnology, Rockford, Ill.,USA)/(Pierce Biotechnology, Rockford, Ill., USA) for one hour at roomtemperature. EDC 5 weight % and NHS 5 weight % aqueous solution wasprepared just before incubation since EDC is unstable in water. Afterone hour the slide was thoroughly washed with water before it wasincubated with 0.1 mg/mL Protein A/G (Pierce Biotechnology, Rockford,Ill., USA) and put in a moist chamber in the cold room over night. Thenext morning, 0.5 mg/mL of biotinylated anti-human ferritin IgG(Rockland Immunochemicals, Inc., Gilbertsville, Pa., USA) in buffer wasspotted onto the glass slide surface and incubated for 30 minutes atroom temperature. 0.5mg/mL of regular anti-human ferritin IgG (RocklandImmunochemicals, Inc., Gilbertsville, Pa., USA) was used as a negativecontrol in this experiment. Three times, the slide was rinsed in 0.1%BSA (Rockland Immunochemicals, Inc., Gilbertsville, Pa., USA) and BSAwas then applied to the whole surface and incubated for 30 minutes atroom temperature. Fluorescein conjugated streptavidin (PierceBiotechnology, Rockford, Ill., USA) was added so that it covered theglass slide and it was incubated for 30 minutes at room temperature. Theslide was scanned at 532 nm (green light) in a GenePix Pro 6.0, 4000AMicroarray Scanner (Axon Instruments Inc., Foster City, Calif., USA).

Example 4 Functionalization of Nanopipettes

After pulling, the nanopipette tips were treated with PLL for fiveminutes and then baked in the oven in 120° C. for one hour. Nanopipettehandling devices, made out of pipette tips and Eppendorf lids, weremounted on a 96-well plate and used for further washings andincubations. Four nanopipettes were mounted in handling devices on a 96well plate. Nanopipettes were dipped into the carboxylated polymer,polyacrylic acid (from Sigma-Aldrich #323667, average Mw˜1,800) for 10minutes and then washed with water three times for at least 30 secondseach wash before they were incubated for an hour in EDC/NHS (50 mg/mLEDC, 50 mg/mL NHS). After washing three times in water for at least 30seconds each wash, the pipettes were put in 0.1 mg/mL Protein A/Gsolution over night in cold room. Next morning, immobilization of aselected antibody (IgG) took place dipping a nanopipette tip in a 40μg/ml IgG solution for 1 hr at room temperature to allow the protein A/Gto capture the Fc region of the IgG molecule. Anti-human VEGF IgG [Goat](R&D Systems, Inc., Minneapolis, USA) or anti-human IL-10 IgG [Rat](BioLegend, San Diego, CA, USA) were used with a concentration of 0.04mg/mL. Anti-human ferritin IgG was used as a negative control with thesame antibody concentration as for the probe nanopipettes. In preparingthe antibody coating, care should be taken to apply antibody primarilyto the tip region. No aspiration was used, but tips are held to lessthan 1 mm insertion into the antibody solution.

Example 5 Antigen and Antibody Interactions

The nanopipette biosensor examples below can be divided into threecategories: I) Primary and secondary antibody interaction experiment,II) Antibody-antigen interaction experiment and III) Limit of detectionexperiment.

I) Primary and Secondary Antibody Interaction Experiment

One primary and secondary antibody interaction experiment was performedand anti-human VEGF IgG [Goat] and anti-human ferritin IgG [Rabbit](control). 10 μL of anti-Goat IgG (Rockland Inc., Gilbertsville, USA)was added twice, with final concentrations of 0.02 mg/mL and 0.04 mg/mL,to the functionalized nanopipette. This approach was not very successfulso a different approach was tried. That is, it was found advantageous tohave the detection antibodies directly coupled to the glass pipette.This approach did not use chemical coupling of the peptide ligand to thenanopipette tip, and was not found to be optimum.

II) Antibody-Antigen Interaction Experiments

Six antibody-antigen interaction experiments were performed.

Two of these were done with an anti-human VEGF IgG [Goat] functionalizednanopipettes adding 10 μL of the antigen, recombinant human VEGF-162(R&D Systems, Inc., Minneapolis, USA) twice, resulting in concentrations0.004 mg/mL and 0.008 mg/mL of VEGF in the nanopipette biosensor bath.The VEGF molecule used consists of 162 amino acid residues with amolecular weight of 18.8 kDa, calculated by R&D Systems. Unlike mostproteins VEGF has a positive net charge.

The other four experiments used anti-human IL-10 IgG functionalizednanopipettes. Recombinant human IL-10 (R&D Systems, Inc., Minneapolis,USA), with a molecular weight of 18.6 kDa, was added to the electrolytesolution with the nanopipettes. In these experiments 10 μL of IL-10solution was added once with a resulting concentration of 0.004 mg/mL.For all the antibody-antigen interaction experiments anti-human ferritinIgG [Rabbit] functionalized nanopipettes were used as a negativecontrol.

III) Limit of Detection Experiment (FIG. 8)

A limit of detection experiment was performed with anti-human IL-10 IgGfunctionalized nanopipettes adding 10 μL recombinant human IL-10resulting in four different concentrations, 4 ng/mL 40ng/mL, 0.4 μg/mLand 4 μg/mL. Also in this experiment an anti-human ferritin IgG [Rabbit]functionalized nanopipette was used as negative control.

Data Analysis

The average current peak values for each sweep, observed on a MolecularDevices Clampfit Screen at ˜1 cycle per second, positive as well asnegative, were collected on a computer and made into a new peak valuegraph to enable relative comparison of current changes between thenanopipette functionalized with probe molecules and the negative controlnanopipette. The peak current data points were normalized against thebaseline upon analyte addition. In this work, a raw data file consistedof multiple sweeps, each sweep being a period, e.g., 50 seconds ofmeasurement of current cycles. During each sweep, 50 cycles ofsinusoidal voltage was applied and corresponding peaks in current weremeasured and averaged. Normalization was done by dividing a givencurrent value by the average immediately before the addition of targetmolecules for the run. As low as 1% change in current can be detected.Normalization methods are also used to facilitate the differentiationbetween random noise of various pipettes resulting from differentfunctionalizations. That is, differently prepared pipettes may havedifferent unblocked currents, e.g., between 1500 and 4000 pA. However,current changes resulting from specific binding can still be detected.Values from different pipettes can be normalized to facilitatecomparisons of results.

There was no apparent difference between the IgG [Goat] functionalizednanopipette and the IgG [Rabbit] (control) functionalized nanopipetteupon addition of anti-goat IgG. Nanopipette measurements in Experiment071206 (See Table below), examining the measurement of interactionsbetween IgG and anti-IgG

Antibody-Antigen Interaction; Anti-human VEGF IgG [Goat] and Human VEGF

Two experiments with anti-human VEGF IgG [Goat] functionalizednanopipettes was carried out. Anti-ferritin IgG [Rabbit] was used formodifying the negative control nanopipette. After adding VEGF twice achange in the negative current profile was seen for the anti-human VEGFIgG [Goat] functionalized nanopipettes in both experiments, indicatingspecific binding, see FIGS. 4-7.

That the change mainly occurred in the anti-human VEGF IgG nanopipettebiosensor was determined. The current response over time was measuredafter normalization by relative comparison with negative control enabledby normalizing the data. In experiment 080112, a change of 10% inpositive current profile and 25% in negative current profile was shown.In the other VEGF experiment the corresponding change was +0 /−20%.

Antibody-Antigen Interaction; Anti-Human IL-10 and Human IL-10

Another antibody-antigen pair was tested after VEGF experimentsindicated specific binding. Four experiments with anti-human IL-10 IgGfunctionalized nanopipettes were performed. One experiment showed asmall current change and one experiment had no significant currentchange upon addition of human IL-10.

Evident current changes of 10% or more were observed in two of theanti-human IL-10 IgG nanopipette biosensor experiments after addinghuman IL-10. As shown in FIG. 9, experiment 080205 showed a large changein the negative ionic current response and a small but significantchange in the positive current response for the anti-human IL-10 IgGfunctionalized nanopipette. The same negative control used in earlierexperiments was used for these experiments.

Table Summarizing Nanopipette Measurement Results

Of the seven specific binding experiments performed four showedsignificant change, one was ambiguous and two remained unchanged.

Normalized Current Change Indication of Specific Experiment(positive/negative) Binding 071206 0 − 080110    0/20-30% + 080112 (FIG.6) 10%/25%  + 080131  ?/5% +/− ? 080205 (FIG. 7) 5%/20% + 080214°2%/10% + 080214b ?/0  −

Nanopipette Biosensor Limit of Detection (LOD)

In an attempt to investigate nanopipette biosensor limit of detectiondifferent amounts of IL-10 were added four times (FIG. 8). The currentprofile changed at the lowest concentration of 4 ng/mL for anti-humanIL-10 IgG nanopipette but also for the negative control. For theanti-human IL-10 IgG functionalized pipette the current change was notas big after a concentration of 0.4 μg/mL IL-10.

Selectivity, Sensitivity and Robustness

Out of the seven experiments performed four indicated specific binding,one had unclear results and two showed no specific binding. Thenanopipette biosensors have repeatedly and with two different cancerbiomarkers as target molecules been shown to be selective but not veryrobust and more experiments need to be performed.

To investigate the nanopipette's sensitivity and the limit of detection(LOD), additional experiments were carried out. In order to ascertainlimit of detection, the first addition of analyte should not havechanged the current, but it did. It was established with this experimentthat a change was only observed for addition to the concentration of 4ng/mL of IL-10 and that the limit of detection for nanopipettebiosensors could be smaller than ˜4 ng/mL. If this is true, thenanopipette biosensor technology is approximately within the samedetection order range as the plasma concentration level of IL-10 incancer patients and could be used for this aim since IL-10 plasmaconcentrations of ˜8 ng/mL have been measured with ELISA forcancer-bearing patients. It should be noted that measuring clean sampleof target protein and target protein in plasma could affect LOD for anytechnique. Current changes upon analyte addition in negative controlwere also seen in this experiment which makes it difficult to draw anyconclusions from these results. A blank addition in the beginningwithout analyte could have helped in the data analysis here.

A trend seen in the nanopipette experiments was that the negative peakcurrent profile changed more than the positive equivalent. Onecontributing cause to this trend may be the ionic permselectivity causedby an electric double layer (EDL) predicted by Wei et al., see FIG. 9.When negative voltage was applied to the nanopipette, a flow ofpotassium ions went through the tip. This ion current will be enhancedif the EDL is also negative. On the other hand, positive applied voltageto the nanopipette tip results in negative chloride ions passing throughthe pore. This flow is smaller due to repelling electrostatic forcesfrom the negatively charged EDL. The EDL is part of a rectificationeffect that functionalization of the nanopipette will create. It isimportant to measure currents in both positive and negative modes.

Given the data presented here, it can be seen that further optimizationof the surface chemistry can be done. Some experiments have suggestedthat cutting incubation time for Protein A/G and antibodies is apossibility. This could further speed up the preparation process. Inaddition, the concentration of immobilized antibodies should beinvestigated more and possibly optimized. For the glass slide experimenta Protein A/G—antibody ratio of 1:5 was used successfully, however dueto quantity constraints the same ratio for the nanopipette experimentswere about 1:0.5.

By using several pipettes in a flow chamber multiplex of the nanopipettebiosensor technology could be made a reality. Serum or other sampleswith multiple analytes could move through the flow chamber and paralleltarget detection by differentiating functionalized nanopipettebiosensors as predicted.

In this example, antibodies were immobilized onto the nanopipettesurface and antigens were added to the electrolyte bath. With adifferent surface chemistry reversed detection could be applied ifantigens were immobilized to the surface and corresponding antibodieswere added to the detection bath. So called antibody sandwich assays,with primary antibody—antigen—secondary antibody binding structure, isalso a plausible approach.

Naturally, nanopipette biosensors are not only applicable to antibodiesand cancer markers but to other probe / target molecules as well.Cytokines with their corresponding receptors are additional interactionpairs to investigate.

In repeated voltage clamp experiments, the nanopipette biosensors provedto specifically detect antigen molecules in real-time at a concentrationof 4 μg/mL. The antigen molecules used were considerably smaller thanantibodies (VEGF and IL-10˜19kDa compared to 150 kDa for mostantibodies), showing that the system is sensitive. In an attempt toestablish limit of detection (LOD), a current profile change wasobserved at a concentration of 4 ng/mL of analyte. Since this was thesmallest concentration used in a nanopipette biosensor experiment sofar, LOD is still unknown but believed to be <4 ng/mL. Compared tostate-of-the-art protein analysis techniques such as ELISA and SPR, thenanopipette biosensor technology platform holds future applicationpotential with further development.

REFERENCES

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CONCLUSION

The above specific description is meant to exemplify and illustrate theinvention and should not be seen as limiting the scope of the invention,which is defined by the literal and equivalent scope of the appendedclaims. Any patents or publications mentioned in this specification areintended to convey details of methods and materials useful in carryingout certain aspects of the invention which may not be explicitly set outbut which would be understood by workers in the field. Such patents orpublications are hereby incorporated by reference to the same extent asif each was specifically and individually incorporated by reference, asneeded for the purpose of describing and enabling the method or materialreferred to.

What is claimed is:
 1. A nanopipette device for specific detection ofone or more analytes in a sample containing electrolyte, comprising: (a)a quartz or glass capillary nanopipette having a tip with a nanoscaleopening between an interior of the nanopipette and an external area forcontacting the sample, said nanopipette having a hollow structuredefining an internal volume communicating with the sample and saidnanoscale opening in the tip of between 10 and 100 nm in diameter; (b)peptide that specifically binds to a predetermined analyte in thesample, said peptide chemically attached to an internal service of saidtip through a polymeric coating, whereby binding of an analyte to thepeptide reduces size of said nanoscale opening; (c) a first electrode,arranged to be in contact with electrolyte in the interior of thenanopipette, and connected to an input of an amplifier comprised in acurrent detecting circuit that detects ionic current through thenanoscale opening; (d) a second electrode, arranged to be in contactwith electrolyte exterior of the nanopipette, and further connected tosaid current detecting circuit, whereby electrolyte in the interior ofthe nanopipette and electrolyte in a bath permits ionic current to flowbetween the first electrode and the second electrode and through thenanoscale opening in the tip; and. (e) said amplifier configured toapply an alternating voltage to the first electrode thereby producingpositive and negative ionic currents, and said current detecting circuitconfigured to detect both of said positive and negative ionic currents.2. The nanopipette device of claim 1 wherein said current detectorcircuit is configured as a voltage clamp in response to an alternatingvoltage signal from a signal generator.
 3. The nanopipette device ofclaim 2 wherein the signal generator comprises an analog to digitalconverter and the alternating voltage signal is sinusoidal.
 4. Thenanopipette device of claim 2 wherein the first electrode is attached toa differential amplifier and the second electrode is attached to areference electrode.
 5. The nanopipette device of claim 1 furthercomprising a plurality of nanopipettes in a single external bath,different nanopipettes having different functionalities, said pluralityof nanopipettes each having therein an electrode connected to an inputof an amplifier comprised in a current detecting circuit that detectsionic current through a nanoscale opening in a member of said pluralityof nanopipettes.
 6. The nanopipette device of claim 1 wherein the firstelectrode and second electrode are separated from each other by filtermaterial between the first electrode and the second electrode, therebypreventing analyte flow between the electrodes but permitting ioniccurrent between the electrodes.
 7. The nanopipette device of claim 1wherein the nanopipette is quartz.
 8. The nanopipette device of claim 1wherein said polymeric coating comprises a layer of carboxylated polymerbonded to said polymeric coating wherein said polymeric coatingcomprises an amine-containing layer bonded to surface of the hollowstructure of the nanopipette.
 9. The nanopipette device of claim 8wherein the carboxylated polymer is polyacrylic acid and theamine-containing layer is poly-1-lysine.
 10. The nanopipette device ofclaim 1 wherein the peptide is chemically attached to the nanopipettethrough binding to a protein comprising protein A , covalently linked tothe polymeric coating on the surface of the hollow structure of thenanopipette.
 11. The nanopipette device of claim 10 comprising a secondnanopipette arranged to contact the sample and comprising a secondpeptide chemically attached to said tip, and further comprising anelectrode within the second nanopipette and arranged to be connected toa second amplifier input in said current detecting circuit.
 12. Thedevice of claim 11 further comprising a filter in the electrolyte forpreventing analyte movement between the first electrode and the secondelectrode.
 13. The nanopipette device of claim 1 wherein the secondpeptide analyte binding molecule is an antibody polypeptide.
 14. Thenanopipette device of claim 1 wherein the peptide is an antibodypolypeptide.
 15. The nanopipette device of claim 1 wherein the peptideis attached to the inside of the nanopipette.
 16. The nanopipette deviceof claim 1 wherein the electrolyte in the bath is an electrolyte gelseparating the first and second electrodes.
 17. The nanopipette deviceof claim 16 wherein the electrolyte gel is agar gel.
 18. The nanopipettedevice of claim 1 wherein the peptide is attached to the nanopipette tipby an NHS (N-hydroxysuccinimide) coupling agent.
 19. The nanopipettedevice of claim 1 wherein said polymeric coating comprises apoly-L-lysine layer.
 20. A method of detecting an analyte in a sample,comprising the steps of: (a) contacting the sample with a nanopipettehaving a tip with a nanoscale opening between an interior of thenanopipette and an external bath, the interior of the nanopipettecommunicating with the tip opening, for containing analyte material andallowing it to pass through the opening, said tip further havingdirectly chemically attached thereto a peptide binding moleculespecifically binding to the analyte; (b) applying an alternating voltageto a first electrode contacting analyte material in the interior of thenanopipette, said electrode being connecting to a current detectingcircuit; (c) measuring ionic current flow between the first electrodeand a second electrode, also for connection to the current detectingcircuit, arranged to be in contact with an electrolyte in the bath,whereby electrolyte in the interior of the nanopipette and electrolytein the bath permits ionic current to flow between the electrodes andthrough the tip, said ionic current being detectibly reduced when thetip is blocked by analyte.
 21. The method of claim 20 wherein thealternating voltage is sinusoidal.
 22. The method of claim 21wherein thealternating voltage alternates at between 0.5 and 10 Hz.
 23. The methodof claim 20 wherein the alternating voltage produces an alternatingionic current that is reduced in both negative amplitude and positivecurrent amplitude upon binding of an analyte.
 24. The method of claim 23wherein the reduction is between 3% and 20%.